Anle138b: a novel oligomer modulator for disease-modifying therapy of neurodegenerative diseases such as prion and Parkinson's disease methods
Aim. Evidence-backed execution summary for Anle138b: a novel oligomer modulator for disease-modifying therapy of neurodegenerative diseases such as prion and Parkinson's disease methods from Anle138b: a novel oligomer modulator for disease-modifying therapy of neurodegenerative diseases such as prion and Parkinson's disease.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Experiments in prion-infected mice
reagent used in the protocol.
- Use
- Seven-week-old female C57BL/6 mice were inoculated intraperitoneally (i.p.) with 100 µl of 1 % (w/v) brain homogenate (RML scrapie). PrP Sc level in the spleen was determined at 35 dpi following 34 days of treatment with 1 mg compound mixed with 10 µl DMSO + 200...
Materials and methods
reagent used in the protocol.
- Use
- The two libraries screened contain 10,000 compounds each and are called DIVERSet1 and DIVERSet2, because they cover only a part of the larger DIVERSet library (ChemBridge Corp., San Diego, CA). DIVERSet is a collection of rationally selected, diverse, drug-like small molecules. The compounds were supplied in dimethy...
Assay for PrP C -PrP Sc association by scanning for intensely fluorescent targets (SIFT)
reagent used in the protocol.
- Use
- Recombinant PrP 23-231 (rPrP) was produced and purified essentially as described [ ]. L42 monoclonal antibody (r-biopharm, Darmstadt, Germany) was labelled with Alexa-Fluor-647 (Invitrogen, Eugene) according to the manufacturer's manual. Recombinant mouse PrP 23-231 was labelled with Alexa-Fluor-488 (Invitroge...
Inhibition of PrP Sc accumulation in a cell-based dot blot assay
reagent used in the protocol.
- Use
- Mouse neuroblastoma (ScN2a) and scrapie mouse brain (SMB) cells (TSE Resource Centre) [ ], infected with the Rocky Mountain Laboratory (RML) scrapie strain, were seeded in a concentration of 20,000 cells per well in 100 µl of medium at a Costar 3599 flat-bottom 96-well plate (Corning Inc., Corning, NY) pri...
Inhibition of PrP Sc accumulation in a cell-based dot blot assay
reagent used in the protocol.
- Use
- After cell medium removal, 100 µl of lysis buffer (50 mM Tris/HCl, ph 8.0, 150 mM NaCl; 0.5 % (w/v) DOC; 0.5 % (v/v) Triton X-100) was added to each well for 5 min at room temperature. Using a dot blot apparatus (Sigma-Aldrich) cell lysate was transferred under vacuum to an activat...
Experiments in prion-infected mice
reagent used in the protocol.
- Use
- For screening and structure-activity analysis, compounds were tested in regard to their inhibitory effect on PrP Sc accumulation in vivo by three experimental protocols: Seven-week-old female C57BL/6 mice were inoculated intracerebrally (i.c.) with 30 µl of 1 % (w/v) brain homogenate (RML scrapi...
Experiments in prion-infected mice
reagent used in the protocol.
- Use
- Seven-week-old female C57BL/6 mice were inoculated i.c. with 30 µl of 1 % (w/v) brain homogenate (RML scrapie). Treatment was started at 80 dpi with 0.84 mg compound (in 25 µl DMSO) per day applied by intraperitoneal injection for 14 days followed by 2 × 5...
Experiments in prion-infected mice
reagent used in the protocol.
- Use
- For long-term survival experiments, anle138b was administered orally in DMSO/peanut butter as described above. In a first set of experiments, 5 mg anle138b were given once daily starting either at day 80 or day 120 post i.c. infection. Animals of each treatment group were monitored daily for signs of disease by...
Assay for PrP C -PrP Sc association by scanning for intensely fluorescent targets (SIFT)
Recombinant PrP 23-231 (rPrP) was produced and purified essentially as described [ ]. L42 monoclonal antibody (r-biopharm, Darmstadt, Germany) was labelled with Alexa-Fluor-647 (Invitrogen, Eugene) according to the manufacturer's manual. Recombinant mouse PrP 23-231 was labelled with Alexa-Fluor-488 (Invitroge...
- Use
- Recombinant PrP 23-231 (rPrP) was produced and purified essentially as described [ ]. L42 monoclonal antibody (r-biopharm, Darmstadt, Germany) was labelled with Alexa-Fluor-647 (Invitrogen, Eugene) according to the manufacturer's manual. Recombinant mouse PrP 23-231 was labelled with Alexa-Fluor-488 (Invitroge...
Inhibition of PrP Sc accumulation in a cell-based dot blot assay
After cell medium removal, 100 µl of lysis buffer (50 mM Tris/HCl, ph 8.0, 150 mM NaCl; 0.5 % (w/v) DOC; 0.5 % (v/v) Triton X-100) was added to each well for 5 min at room temperature. Using a dot blot apparatus (Sigma-Aldrich) cell lysate was transferred under vacuum to an activat...
- Use
- After cell medium removal, 100 µl of lysis buffer (50 mM Tris/HCl, ph 8.0, 150 mM NaCl; 0.5 % (w/v) DOC; 0.5 % (v/v) Triton X-100) was added to each well for 5 min at room temperature. Using a dot blot apparatus (Sigma-Aldrich) cell lysate was transferred under vacuum to an activat...
Histology and immunohistochemistry
Prion infectivity was inactivated by immersion in 100 % formic acid [ ]. Paraffin-fixed sections (2.5 µm) of brain tissue were stained with H&E. For PrP Sc detection using antibody CDC1 [ ], sections were immunostained on a Ventana automated staining apparatus. To assess neuronal degeneration, neurons...
- Use
- Prion infectivity was inactivated by immersion in 100 % formic acid [ ]. Paraffin-fixed sections (2.5 µm) of brain tissue were stained with H&E. For PrP Sc detection using antibody CDC1 [ ], sections were immunostained on a Ventana automated staining apparatus. To assess neuronal degeneration, neurons...
Quantification of PrP Sc
For quantification of PrP Sc, brain homogenates were homogenized 10 % (w/v) in lysis buffer (100 mM NaCl, 10 mM EDTA, 0.5 % (v/v) NP-40, 0.5 % (w/v) deoxycholate, 10 mM Tris/HCl pH 7.4) and subjected to dot blot analysis using 6H4 antibody (Prionics, Switzerland) at a dilution of 1:5,0...
- Use
- For quantification of PrP Sc, brain homogenates were homogenized 10 % (w/v) in lysis buffer (100 mM NaCl, 10 mM EDTA, 0.5 % (v/v) NP-40, 0.5 % (w/v) deoxycholate, 10 mM Tris/HCl pH 7.4) and subjected to dot blot analysis using 6H4 antibody (Prionics, Switzerland) at a dilution of 1:5,0...
Protein misfolding cyclic amplification (PMCA) [, ]
PMCA product was digested with 50 µg/ml proteinase K at 37 °C for 1 h. After adding an equal volume of SDS loading buffer and boiling for 10 min, samples were separated by SDS-PAGE and transferred to PVDF membranes (Immobilon-P, Millipore, MA, USA) at 12 V for 2 h. For immunob...
- Use
- PMCA product was digested with 50 µg/ml proteinase K at 37 °C for 1 h. After adding an equal volume of SDS loading buffer and boiling for 10 min, samples were separated by SDS-PAGE and transferred to PVDF membranes (Immobilon-P, Millipore, MA, USA) at 12 V for 2 h. For immunob...
α-Synuclein in vitro oligomer formation assay
Aggregation of α-syn was measured by scanning for intensely fluorescent targets (SIFT) [ ]. SIFT measurements were carried out on an Insight Reader (Evotec-Technologies) with dual-colour excitation at 488 and 633 nm, using a 40 × 1.2 numerical aperture microscope objective (Olympus, Japan) a...
- Use
- Aggregation of α-syn was measured by scanning for intensely fluorescent targets (SIFT) [ ]. SIFT measurements were carried out on an Insight Reader (Evotec-Technologies) with dual-colour excitation at 488 and 633 nm, using a 40 × 1.2 numerical aperture microscope objective (Olympus, Japan) a...
α-syn oligomer pore formation in planar lipid bilayers
Planar lipid bilayers were generated and analyzed in a Ionovation Compact (Ionovation, Osnabrück, Germany) by the painting technique [ ]. Two bath chambers (1.2 ml) separated by a Teflon-septum were filled with 250 mM KCl, 10 mM MOPS, pH = 7.2 (Merck, Darmstadt, Germany and Roth, Karlsr...
- Use
- Planar lipid bilayers were generated and analyzed in a Ionovation Compact (Ionovation, Osnabrück, Germany) by the painting technique [ ]. Two bath chambers (1.2 ml) separated by a Teflon-septum were filled with 250 mM KCl, 10 mM MOPS, pH = 7.2 (Merck, Darmstadt, Germany and Roth, Karlsr...
Oral rotenone in vivo mouse model of Parkinson's disease
One-year old C57Bl/6J mice were maintained in a constant light/dark cycle (12 h/12 h). Water and food were given ad libitum. Mice were divided into 4 groups ("no rotenone", "rotenone/normal feed", "rotenone/placebo feed", "rotenone/anle138b"). A further group...
- Use
- One-year old C57Bl/6J mice were maintained in a constant light/dark cycle (12 h/12 h). Water and food were given ad libitum. Mice were divided into 4 groups ("no rotenone", "rotenone/normal feed", "rotenone/placebo feed", "rotenone/anle138b"). A further group...
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Inhibition of PrP Sc accumulation in a cell-based dot blot assay
After cell medium removal, 100 µl of lysis buffer (50 mM Tris/HCl, ph 8.0, 150 mM NaCl; 0.5 % (w/v) DOC; 0.5 % (v/v) Triton X-100) was added to each well for 5 min at room temperature. Using a dot blot apparatus (Sigma-Aldrich) cell lysate was transferred under vacuum to an activated polyvinylidene difluoride (PVDF) membrane (Immobilon-P; Milipore) and fixed to the membrane by incubation for 1 h at 37 °C. The membrane was incubated in lysis buffer and treated with proteinase K solution (final dilution 25 µg/ml) for 90 min at 37 °C. Subsequently, the membrane was washed twice with pure water, the denaturation solution (3 M Guanidiniumthiocyanat, 0.1 M TrisHCl pH 8.0) was added for 15 min at room temperature and membranes were washed five times with pure water. After the denaturation step the membr...
Experiments in prion-infected mice
For long-term survival experiments, anle138b was administered orally in DMSO/peanut butter as described above. In a first set of experiments, 5 mg anle138b were given once daily starting either at day 80 or day 120 post i.c. infection. Animals of each treatment group were monitored daily for signs of disease by trained animal caretakers from day 80 post infection. The animals were sacrificed, when they had reached the terminal stage of the disease based on clinical signs (ataxia, tremor, difficulty in righting up from a position lying on its back and tail stiffness). Typically the disease progress through the terminal stage will lead to the death of the animal within 1 or 2 days. In addition, groups of four mice per experimental group were sacrificed at predefined time points as indicated in Fig. c-e. From all animals, one brain hemisphere and one half of the spl...
Histology and immunohistochemistry
Prion infectivity was inactivated by immersion in 100 % formic acid [ ]. Paraffin-fixed sections (2.5 µm) of brain tissue were stained with H&E. For PrP Sc detection using antibody CDC1 [ ], sections were immunostained on a Ventana automated staining apparatus. To assess neuronal degeneration, neurons with pyknotic nuclei were counted in blinded slides in the cerebellar granule cell layer. This approach has been validated previously as an efficient measure for apoptotic neuronal cell death [ ]. For each animal, 30 randomly chosen high power visual fields were analyzed.
Protein misfolding cyclic amplification (PMCA) [, ]
PMCA was performed in a water-bath sonicator (Misonix sonicator 3000, Misonix, Farmingdale, NY, USA), which had a microplate horn for PCR tubes. Normal brain homogenate was mixed with infected brain homogenate (100:1 v/v) and 99 µl of this mixture was transferred into 0.2-ml PCR tubes which contained 1 µl of DMSO or compound solved in DMSO. For mouse substrates 18 PMCA cycles and for human substrates 40 cycles were performed. Each cycle consisted of sonication at 60 % potency (~209 W) for 20 s followed by incubation at 37 °C for 59 min 40 s.
Protein misfolding cyclic amplification (PMCA) [, ]
PMCA product was digested with 50 µg/ml proteinase K at 37 °C for 1 h. After adding an equal volume of SDS loading buffer and boiling for 10 min, samples were separated by SDS-PAGE and transferred to PVDF membranes (Immobilon-P, Millipore, MA, USA) at 12 V for 2 h. For immunoblotting, the membrane was blocked with 5 % non-fat milk in PBST and incubated with 1:5,000 diluted PrP monoclonal antibody 3F4 (Dako, Glostrup, Denmark) in PBST at room temperature for 2 h. After three washes in PBST, the membrane was immerged in a 1:5,000 diluted alkaline phosphatase conjugated anti-mouse IgG (Dako) in PBST and incubated at room temperature for 2 h. Detection was performed with CDP-Star solution (Roche, Mannheim, Germany). Immunoblots were scanned and quantified by a Diana III luminescence imaging system along with the AIDA software package...
α-Synuclein in vitro oligomer formation assay
The α-syn aggregation assay was conducted with minor modifications as published previously [ ]. All experiments were performed in 50 mM Tris-buffer (pH 7.0) in a total volume of 20 µl in 384- or 96-well plates with a cover slide bottom (Evotec-Technologies, Germany). The concentration of α-syn was approximately 20 nM. In all experiments, α-syn aggregation was induced by final concentrations of 1 % DMSO and 10 µM FeCl 3. Compounds were diluted into the assay mixture from 10-fold stock solutions containing 10 % DMSO (v/v), resulting in a final concentration of 1 % DMSO in all samples. "Low control" measurements for threshold setting were obtained without FeCl 3 and DMSO. Experiments were started by diluting the 5-fold stock solution of fluorescently labelled α-syn in 50 mM Tris-buffer, pH 7.0, containing...
α-syn oligomer pore formation in planar lipid bilayers
Planar lipid bilayers were generated and analyzed in a Ionovation Compact (Ionovation, Osnabrück, Germany) by the painting technique [ ]. Two bath chambers (1.2 ml) separated by a Teflon-septum were filled with 250 mM KCl, 10 mM MOPS, pH = 7.2 (Merck, Darmstadt, Germany and Roth, Karlsruhe, Germany). Then, 2 µl of a 100 mg/ml-solution of purified azolectin in n -Decan (Ionovation, Osnabrück, Germany) were applied to a 120-µm pinhole in the septum. After 30 min incubation at room temperature, lipid was thinned out by repetitive lowering and re-raising of the buffer level until a bilayer was formed. This formation was monitored optically and by capacitance measurements. The resulting bilayers had a typical capacitance of 60-80 pF and a resistance of >100 GΩ. Measurements were performed using Ag/AgCl-electr...
Transgenic α-synuclein mouse model
The in vivo effect of anle138b was tested in a well-established murine α-synucleinopathy model {(Thy1)-h[A30P]α-syn} on a genetic background of C57/Bl6 mice [ ]. Anle138b treatment was tested against placebo treatment with the vehicle (DMSO/peanut butter). Furthermore, untreated non-transgenic C57/Bl6 mice were examined as age-matched controls. The animals were evaluated clinically regarding survival time, motor performance and body weight. In addition, we conducted histological post mortem evaluation of α-syn deposition. Treatment with anle138b and placebo, respectively, was initiated at the age of 8 weeks. Transgenic animals in the treatment and placebo group were matched both in regard to litter and sex. Anle138b was solved in DMSO and mixed with peanut butter. Five days prior to the first dose of anle138b, initial doses of 200 µl peanut butter (Barney...
Measurement outputs
What raw and processed outputs should exist?
The two libraries screened contain 10,000 compounds each and are called DIVERSet1 and DIVERSet2, because they cover only a part of the larger DIVERSet library (ChemBridge Corp.,...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
After cell medium removal, 100 µl of lysis buffer (50 mM Tris/HCl, ph 8.0, 150 mM NaCl; 0.5 % (w/v) DOC; 0.5 % (v/v) Triton X-100) was added to eac...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
For screening and structure-activity analysis, compounds were tested in regard to their inhibitory effect on PrP Sc accumulation in vivo by three experimental protocols: S...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
For long-term survival experiments, anle138b was administered orally in DMSO/peanut butter as described above. In a first set of experiments, 5 mg anle138b were given once...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect the raw assay or blot output and retain identifiers for each sample and experimental group.
inferred from protocolPreprocessing / cleaning
The two libraries screened contain 10,000 compounds each and are called DIVERSet1 and DIVERSet2, because they cover only a part of the larger DIVERSet library (ChemBridge Corp., San Diego, CA).
from paperScoring or quantification
Quantify the primary readouts for this experiment: The two libraries screened contain 10,000 compounds each and are called DIVERSet1 and DIVERSet2, because they cover only a part of the larger DIVERSet library (ChemBridge Corp.,...; After cell medium removal, 100 µl of lysis buffer (50 mM Tris/HCl, ph 8.0, 150 mM NaCl; 0.5 % (w/v) DOC; 0.5 % (v/v) Triton X-100) was added to eac...; For screening and structure-activity analysis, compounds were tested in regard to their inhibitory effect on PrP Sc accumulation in vivo by three experimental protocols: S...; For long-term survival experiments, anle138b was administered orally in DMSO/peanut butter as described above. In a first set of experiments, 5 mg anle138b were given once....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
The two libraries screened contain 10,000 compounds each and are called DIVERSet1 and DIVERSet2, because they cover only a part of the larger DIVERSet library (ChemBridge Corp.,...; SAR was primarily investigated through the effect of treatment for 40 days [80-120 days post infection (dpi)] on PrP Sc accumulation, which provides a rapid and...; Regarding mode of action, these findings in combination with the finding that PrP C expression is unaffected by anle138b (Suppl. Fig. 7) indicate that anle138b directly blo...; α-Synucleinopathies share molecular features in regard to aggregate structure and seeding with prion diseases [,, ] and are of high clinical importance due to their high...
from paperReporting output
Report representative outputs alongside summary comparisons for The two libraries screened contain 10,000 compounds each and are called DIVERSet1 and DIVERSet2, because they cover only a part of the larger DIVERSet library (ChemBridge Corp.,..., After cell medium removal, 100 µl of lysis buffer (50 mM Tris/HCl, ph 8.0, 150 mM NaCl; 0.5 % (w/v) DOC; 0.5 % (v/v) Triton X-100) was added to eac..., For screening and structure-activity analysis, compounds were tested in regard to their inhibitory effect on PrP Sc accumulation in vivo by three experimental protocols: S..., For long-term survival experiments, anle138b was administered orally in DMSO/peanut butter as described above. In a first set of experiments, 5 mg anle138b were given once....
inferred from protocolStructured statistical methods
The two libraries screened contain 10,000 compounds each and are called DIVERSet1 and DIVERSet2, because they cover only a part of the larger DIVERSet library (ChemBridge Corp.,...; SAR was primarily investigated through the effect of treatment for 40 days [80-120 days post infection (dpi)] on PrP Sc accumulation, which provides a rapid and...; Regarding mode of action, these findings in combination with the finding that PrP C expression is unaffected by anle138b (Suppl. Fig. 7) indicate that anle138b directly blo...; α-Synucleinopathies share molecular features in regard to aggregate structure and seeding with prion diseases [,, ] and are of high clinical importance due to their high...
source structuredSource and audit
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Evidence quotes (8)
After cell medium removal, 100 µl of lysis buffer (50 mM Tris/HCl, ph 8.0, 150 mM NaCl; 0.5 % (w/v) DOC; 0.5 % (v/v) Triton X-100) was added to each well for 5 min at room temperature. Using a dot blot apparatus (Sigma-Aldrich) cell lysate was transferred under vacuum to an activated polyvinylidene difluoride (PVDF) membrane (Immobilon-P; Milipore) and fixed to the membrane by incubation for 1 h at 37 °C. The membrane was incubated in lysis buffer and treated with proteinase K solution (final dilution 25 µg/ml) for 90 min at 37 °C. Subsequently, the membrane was washed twice with pure water, the denaturation solution (3 M Guanidiniumthiocyanat, 0.1 M TrisHCl pH 8.0) was added for 15 min at room temperature and membranes were washed five times with pure water. After the denaturation step the membrane was blocked with PBST-milk [5 % (w/v) non-fat milk, 0.1 % (v/v) tween-20 (Sigma) in phosphate buffered saline (PBS)] for 60 min. An appropriate dilution of polyclonal antibody "rabbit-10" [ ] in PBST-milk was incubated with the membrane for 60 min. After PBST rins...
For long-term survival experiments, anle138b was administered orally in DMSO/peanut butter as described above. In a first set of experiments, 5 mg anle138b were given once daily starting either at day 80 or day 120 post i.c. infection. Animals of each treatment group were monitored daily for signs of disease by trained animal caretakers from day 80 post infection. The animals were sacrificed, when they had reached the terminal stage of the disease based on clinical signs (ataxia, tremor, difficulty in righting up from a position lying on its back and tail stiffness). Typically the disease progress through the terminal stage will lead to the death of the animal within 1 or 2 days. In addition, groups of four mice per experimental group were sacrificed at predefined time points as indicated in Fig. c-e. From all animals, one brain hemisphere and one half of the spleen were freshly frozen at -80 °C for biochemical analysis. The other hemisphere and the remaining half of the spleen as well as all inner organs were fixed in 4 % formaldehyde solution for histological analysis. In a further experiment, treatment with anle138b was started on th...
Prion infectivity was inactivated by immersion in 100 % formic acid [ ]. Paraffin-fixed sections (2.5 µm) of brain tissue were stained with H&E. For PrP Sc detection using antibody CDC1 [ ], sections were immunostained on a Ventana automated staining apparatus. To assess neuronal degeneration, neurons with pyknotic nuclei were counted in blinded slides in the cerebellar granule cell layer. This approach has been validated previously as an efficient measure for apoptotic neuronal cell death [ ]. For each animal, 30 randomly chosen high power visual fields were analyzed.
PMCA was performed in a water-bath sonicator (Misonix sonicator 3000, Misonix, Farmingdale, NY, USA), which had a microplate horn for PCR tubes. Normal brain homogenate was mixed with infected brain homogenate (100:1 v/v) and 99 µl of this mixture was transferred into 0.2-ml PCR tubes which contained 1 µl of DMSO or compound solved in DMSO. For mouse substrates 18 PMCA cycles and for human substrates 40 cycles were performed. Each cycle consisted of sonication at 60 % potency (~209 W) for 20 s followed by incubation at 37 °C for 59 min 40 s.
PMCA product was digested with 50 µg/ml proteinase K at 37 °C for 1 h. After adding an equal volume of SDS loading buffer and boiling for 10 min, samples were separated by SDS-PAGE and transferred to PVDF membranes (Immobilon-P, Millipore, MA, USA) at 12 V for 2 h. For immunoblotting, the membrane was blocked with 5 % non-fat milk in PBST and incubated with 1:5,000 diluted PrP monoclonal antibody 3F4 (Dako, Glostrup, Denmark) in PBST at room temperature for 2 h. After three washes in PBST, the membrane was immerged in a 1:5,000 diluted alkaline phosphatase conjugated anti-mouse IgG (Dako) in PBST and incubated at room temperature for 2 h. Detection was performed with CDP-Star solution (Roche, Mannheim, Germany). Immunoblots were scanned and quantified by a Diana III luminescence imaging system along with the AIDA software package (Raytest, Straubenhardt, Germany). In the Western blots shown in Fig. c, d "start" indicates the sample taken from the PMCA reaction mixture at time point 0 after mixing normal brain homogenate with a minute amount of PrP Sc that acts as a seed for the subsequent PMCA reaction. Thu...
The α-syn aggregation assay was conducted with minor modifications as published previously [ ]. All experiments were performed in 50 mM Tris-buffer (pH 7.0) in a total volume of 20 µl in 384- or 96-well plates with a cover slide bottom (Evotec-Technologies, Germany). The concentration of α-syn was approximately 20 nM. In all experiments, α-syn aggregation was induced by final concentrations of 1 % DMSO and 10 µM FeCl 3. Compounds were diluted into the assay mixture from 10-fold stock solutions containing 10 % DMSO (v/v), resulting in a final concentration of 1 % DMSO in all samples. "Low control" measurements for threshold setting were obtained without FeCl 3 and DMSO. Experiments were started by diluting the 5-fold stock solution of fluorescently labelled α-syn in 50 mM Tris-buffer, pH 7.0, containing 1 % DMSO, 10 µM FeCl 3 and compounds in concentrations ranging from 1-10 µM. Aggregation was monitored at room temperature for at least 2.5 h in 3-4 independent samples for each experimental group.
Planar lipid bilayers were generated and analyzed in a Ionovation Compact (Ionovation, Osnabrück, Germany) by the painting technique [ ]. Two bath chambers (1.2 ml) separated by a Teflon-septum were filled with 250 mM KCl, 10 mM MOPS, pH = 7.2 (Merck, Darmstadt, Germany and Roth, Karlsruhe, Germany). Then, 2 µl of a 100 mg/ml-solution of purified azolectin in n -Decan (Ionovation, Osnabrück, Germany) were applied to a 120-µm pinhole in the septum. After 30 min incubation at room temperature, lipid was thinned out by repetitive lowering and re-raising of the buffer level until a bilayer was formed. This formation was monitored optically and by capacitance measurements. The resulting bilayers had a typical capacitance of 60-80 pF and a resistance of >100 GΩ. Measurements were performed using Ag/AgCl-electrodes connected to an EPC10-amplifier and Patchmaster software (HEKA, Lambrecht/Pfalz, Germany). The noise was ~0.5 pA at 3 kHz bandwidth. α-syn was incubated in 50 mM Tris-HCl, pH 7.0 with 1 % DMSO (Sigma-Aldrich, Taufkirchen, Germany) and 20 µM FeCl 3 (Merck...
The in vivo effect of anle138b was tested in a well-established murine α-synucleinopathy model {(Thy1)-h[A30P]α-syn} on a genetic background of C57/Bl6 mice [ ]. Anle138b treatment was tested against placebo treatment with the vehicle (DMSO/peanut butter). Furthermore, untreated non-transgenic C57/Bl6 mice were examined as age-matched controls. The animals were evaluated clinically regarding survival time, motor performance and body weight. In addition, we conducted histological post mortem evaluation of α-syn deposition. Treatment with anle138b and placebo, respectively, was initiated at the age of 8 weeks. Transgenic animals in the treatment and placebo group were matched both in regard to litter and sex. Anle138b was solved in DMSO and mixed with peanut butter. Five days prior to the first dose of anle138b, initial doses of 200 µl peanut butter (Barney′s best creamy peanut butter, Dockhorn & Co, Hamburg, Germany) were given to the mice once daily. During the first 2 weeks of treatment, 2 mg of anle138b dissolved in 10 µl DMSO mixed with 200 µl peanut butter were given. After 2 weeks of treatment, the dose...
Machine-readable layer
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"name": "Anle138b: a novel oligomer modulator for disease-modifying therapy of neurodegenerative diseases such as prion and Parkinson's disease methods",
"description": "Evidence-backed execution summary for Anle138b: a novel oligomer modulator for disease-modifying therapy of neurodegenerative diseases such as prion and Parkinson's disease methods from Anle138b: a novel oligomer modulator for disease-modifying therapy of neurodegenerative diseases such as prion and Parkinson's disease.",
"step": [
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"name": "Inhibition of PrP Sc accumulation in a cell-based dot blot assay",
"text": "After cell medium removal, 100 µl of lysis buffer (50 mM Tris/HCl, ph 8.0, 150 mM NaCl; 0.5 % (w/v) DOC; 0.5 % (v/v) Triton X-100) was added to each well for 5 min at room temperature. Using a dot blot apparatus (Sigma-Aldrich) cell lysate was transferred under vacuum to an activated polyvinylidene difluoride (PVDF) membrane (Immobilon-P; Milipore) and fixed to the membrane by incubation for 1 h at 37 °C. The membrane was incubated in lysis buffer and treated with proteinase K solution (final dilution 25 µg/ml) for 90 min at 37 °C. Subsequently, the membrane was washed twice with pure water, the denaturation solution (3 M Guanidiniumthiocyanat, 0.1 M TrisHCl pH 8.0) was added for 15 min at room temperature and membranes were washed five times with pure water. After the denaturation step the membr..."
},
{
"@type": "HowToStep",
"position": 2,
"name": "Experiments in prion-infected mice",
"text": "For long-term survival experiments, anle138b was administered orally in DMSO/peanut butter as described above. In a first set of experiments, 5 mg anle138b were given once daily starting either at day 80 or day 120 post i.c. infection. Animals of each treatment group were monitored daily for signs of disease by trained animal caretakers from day 80 post infection. The animals were sacrificed, when they had reached the terminal stage of the disease based on clinical signs (ataxia, tremor, difficulty in righting up from a position lying on its back and tail stiffness). Typically the disease progress through the terminal stage will lead to the death of the animal within 1 or 2 days. In addition, groups of four mice per experimental group were sacrificed at predefined time points as indicated in Fig. c-e. From all animals, one brain hemisphere and one half of the spl..."
},
{
"@type": "HowToStep",
"position": 3,
"name": "Histology and immunohistochemistry",
"text": "Prion infectivity was inactivated by immersion in 100 % formic acid [ ]. Paraffin-fixed sections (2.5 µm) of brain tissue were stained with H&E. For PrP Sc detection using antibody CDC1 [ ], sections were immunostained on a Ventana automated staining apparatus. To assess neuronal degeneration, neurons with pyknotic nuclei were counted in blinded slides in the cerebellar granule cell layer. This approach has been validated previously as an efficient measure for apoptotic neuronal cell death [ ]. For each animal, 30 randomly chosen high power visual fields were analyzed."
},
{
"@type": "HowToStep",
"position": 4,
"name": "Protein misfolding cyclic amplification (PMCA) [, ]",
"text": "PMCA was performed in a water-bath sonicator (Misonix sonicator 3000, Misonix, Farmingdale, NY, USA), which had a microplate horn for PCR tubes. Normal brain homogenate was mixed with infected brain homogenate (100:1 v/v) and 99 µl of this mixture was transferred into 0.2-ml PCR tubes which contained 1 µl of DMSO or compound solved in DMSO. For mouse substrates 18 PMCA cycles and for human substrates 40 cycles were performed. Each cycle consisted of sonication at 60 % potency (~209 W) for 20 s followed by incubation at 37 °C for 59 min 40 s."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Protein misfolding cyclic amplification (PMCA) [, ]",
"text": "PMCA product was digested with 50 µg/ml proteinase K at 37 °C for 1 h. After adding an equal volume of SDS loading buffer and boiling for 10 min, samples were separated by SDS-PAGE and transferred to PVDF membranes (Immobilon-P, Millipore, MA, USA) at 12 V for 2 h. For immunoblotting, the membrane was blocked with 5 % non-fat milk in PBST and incubated with 1:5,000 diluted PrP monoclonal antibody 3F4 (Dako, Glostrup, Denmark) in PBST at room temperature for 2 h. After three washes in PBST, the membrane was immerged in a 1:5,000 diluted alkaline phosphatase conjugated anti-mouse IgG (Dako) in PBST and incubated at room temperature for 2 h. Detection was performed with CDP-Star solution (Roche, Mannheim, Germany). Immunoblots were scanned and quantified by a Diana III luminescence imaging system along with the AIDA software package..."
},
{
"@type": "HowToStep",
"position": 6,
"name": "α-Synuclein in vitro oligomer formation assay",
"text": "The α-syn aggregation assay was conducted with minor modifications as published previously [ ]. All experiments were performed in 50 mM Tris-buffer (pH 7.0) in a total volume of 20 µl in 384- or 96-well plates with a cover slide bottom (Evotec-Technologies, Germany). The concentration of α-syn was approximately 20 nM. In all experiments, α-syn aggregation was induced by final concentrations of 1 % DMSO and 10 µM FeCl 3. Compounds were diluted into the assay mixture from 10-fold stock solutions containing 10 % DMSO (v/v), resulting in a final concentration of 1 % DMSO in all samples. \"Low control\" measurements for threshold setting were obtained without FeCl 3 and DMSO. Experiments were started by diluting the 5-fold stock solution of fluorescently labelled α-syn in 50 mM Tris-buffer, pH 7.0, containing..."
},
{
"@type": "HowToStep",
"position": 7,
"name": "α-syn oligomer pore formation in planar lipid bilayers",
"text": "Planar lipid bilayers were generated and analyzed in a Ionovation Compact (Ionovation, Osnabrück, Germany) by the painting technique [ ]. Two bath chambers (1.2 ml) separated by a Teflon-septum were filled with 250 mM KCl, 10 mM MOPS, pH = 7.2 (Merck, Darmstadt, Germany and Roth, Karlsruhe, Germany). Then, 2 µl of a 100 mg/ml-solution of purified azolectin in n -Decan (Ionovation, Osnabrück, Germany) were applied to a 120-µm pinhole in the septum. After 30 min incubation at room temperature, lipid was thinned out by repetitive lowering and re-raising of the buffer level until a bilayer was formed. This formation was monitored optically and by capacitance measurements. The resulting bilayers had a typical capacitance of 60-80 pF and a resistance of >100 GΩ. Measurements were performed using Ag/AgCl-electr..."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Transgenic α-synuclein mouse model",
"text": "The in vivo effect of anle138b was tested in a well-established murine α-synucleinopathy model {(Thy1)-h[A30P]α-syn} on a genetic background of C57/Bl6 mice [ ]. Anle138b treatment was tested against placebo treatment with the vehicle (DMSO/peanut butter). Furthermore, untreated non-transgenic C57/Bl6 mice were examined as age-matched controls. The animals were evaluated clinically regarding survival time, motor performance and body weight. In addition, we conducted histological post mortem evaluation of α-syn deposition. Treatment with anle138b and placebo, respectively, was initiated at the age of 8 weeks. Transgenic animals in the treatment and placebo group were matched both in regard to litter and sex. Anle138b was solved in DMSO and mixed with peanut butter. Five days prior to the first dose of anle138b, initial doses of 200 µl peanut butter (Barney..."
}
],
"tool": [
{
"@type": "HowToTool",
"name": "Assay for PrP C -PrP Sc association by scanning for intensely fluorescent targets (SIFT)"
},
{
"@type": "HowToTool",
"name": "Inhibition of PrP Sc accumulation in a cell-based dot blot assay"
},
{
"@type": "HowToTool",
"name": "Histology and immunohistochemistry"
},
{
"@type": "HowToTool",
"name": "Quantification of PrP Sc"
},
{
"@type": "HowToTool",
"name": "Protein misfolding cyclic amplification (PMCA) [, ]"
},
{
"@type": "HowToTool",
"name": "α-Synuclein in vitro oligomer formation assay"
},
{
"@type": "HowToTool",
"name": "α-syn oligomer pore formation in planar lipid bilayers"
},
{
"@type": "HowToTool",
"name": "Oral rotenone in vivo mouse model of Parkinson's disease"
}
],
"supply": [
{
"@type": "HowToSupply",
"name": "Experiments in prion-infected mice"
},
{
"@type": "HowToSupply",
"name": "Materials and methods"
},
{
"@type": "HowToSupply",
"name": "Assay for PrP C -PrP Sc association by scanning for intensely fluorescent targets (SIFT)"
},
{
"@type": "HowToSupply",
"name": "Inhibition of PrP Sc accumulation in a cell-based dot blot assay"
},
{
"@type": "HowToSupply",
"name": "Inhibition of PrP Sc accumulation in a cell-based dot blot assay"
},
{
"@type": "HowToSupply",
"name": "Experiments in prion-infected mice"
},
{
"@type": "HowToSupply",
"name": "Experiments in prion-infected mice"
},
{
"@type": "HowToSupply",
"name": "Experiments in prion-infected mice"
}
],
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"headline": "Anle138b: a novel oligomer modulator for disease-modifying therapy of neurodegenerative diseases such as prion and Parkinson's disease",
"datePublished": "2013",
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"name": "Johannes Levin"
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"name": "Song Shi"
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"name": "Felix Schmidt"
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"name": "Catharina Prix"
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"@type": "Person",
"name": "Jens Pilger"
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"@type": "Person",
"name": "Markus Zweckstetter"
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"name": "Tobias Frank"
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"name": "Mathias Bähr"
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"name": "Jochen H. Weishaupt"
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"name": "Manfred Uhr"
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